Topic 7 - modern genetics

Question 1

what doe PCR stand for?

Answer 1

polymerase chain reaction

Question 2

what is a PCR used for?

Answer 2

To amplify DNA (make lots of copies of the sample)

Question 3

What are the steps of a PCR?

Answer 3

denaturation:
heat DNA to 95 degrees
this splits strands by breaking h bonds
use enzyme from taq (can withstand heat)

annealing:
cool DNA to 55 degrees, primers bind to strands

extension:
heat to 72 degrees
add one polymerase strand to synthesise DNA from where the primer attached

cycle is repeated around 30 times to give enough DNA. sample can be used in DNA sequencing and DNA profiling.

Question 4

what machine does a PCR?

Answer 4

thermocycler

Question 5

what is a genome?

Answer 5

total genetic makeup of an organism

Question 6

what are introns/exons

Answer 6

introns- nucleotide sequences that DON’T code for a gene

exons- nucleotide sequences DO code for a gene

Question 7

why is a pH buffer used in PCR?

Answer 7

to keep the pH constant and maintain optimum pH level for enzyme activity

Question 8

what is an intron?

Answer 8

DNA that does not code for a protein

Question 9

within introns , there are satellites (STRs) - what are these?

Answer 9

satellites are found within introns , they are short sequences of DNA that are repeated many times.

Question 10

what are the two types of satellites (STRs) ?

Answer 10

micro satellites- 2-6 bases repeated 5-100 times

mini satellites 10-100 bases, repeated 50-several hundred times

Question 11

How do satellites (STRs) determine how closely related individuals are?

Answer 11

in a homologous pair of chromosomes, the same satellite is found at the same location - the thing that makes us unique is how often this is repeated.

The more closely related, the more similar the patternss in the size of satellites.

Question 12

What are the stages of DNA profiling?

Answer 12

PCR happens previously.

1. RESTRICTION ENDONUCLEASE ENZYMES
   used to cut DNA as they are made to have a specific active site which fits onto the specific intron where it wants to cut
2. EITHER SIDE OF SATELLITE IS CUT
3. GEL ELECTROPHORESIS
4. SOUTHERN BLOTTING
   alkaline buffer added (denatures DNA so it becomes single stranded)
   Nylon filler placed on gel- this draws the DNA fragments to it leaving them as ‘blots’ attached.
5. ADD GENE PROBES
   Gene probes are short single strand of DNA that have sequences complementary t sequences being sought.
   Probes have fluorescent marker attached
   bind with complementary DNA strands in a process called - hybridisation
6. GRAPH THE DATE COLLECTED FROM THE PROBE

Question 13

What is the name of the process when cells become specialised?

Answer 13

differentiation

Question 14

How do cells become specialised?

Answer 14

the genes we don’t want are switched off.

This means transcription doesn’t happen so RNA is not made , so gene is not translated.

Question 15

what are house keeping proteins?

Answer 15

housekeeping proteins are proteins present in most cells , for example ATP synthase

you can compare between cells by comparing the RNAs (they all have same DNA)

Question 16

what is a regulatory sequence and where is it found?

Answer 16

a regulatory sequence determines whether a gene is expressed or not -> these are found in our ‘junk DNA’ which consists of non-coding introns

regulatory sequences are either promoter or enhancer regions

Question 17

a regulatory sequence can be either a promotor or enhancer region

what does a promotor region do?

Answer 17

initiate transcription by enabling RNA polymerase to bind to the region they regulate

Question 18

a regulatory sequence can be either a promotor or enhancer region

what does an enhancer region do?

Answer 18

makes the gene more/less accessible by opening the chromatin structure (by loosening or tightening DNA around histome (when tightened, no gene)

Question 19

what is a transcription factor?

Answer 19

Transcription factors are proteins that bind to DNA. They either stimulate/prevent transcription of the gene.

They can either be:

promotor sequences- enable binding of RNA polymerase and therefore enable transcription

enhancer sequences- regulate DNA activity by changing chromatin structure. If structure open, gene is expressed. If closed, gene inactivity.

Question 20

explain how splicing happens

Answer 20

splicing is post-transcriptional modification of the mRNA. Helps explain how cells produce more proteins than they have genes, because it results in different products from a single gene.

happens during transcription.

full section of DNA copied onto mRNA (now known as pre-mRNA)

splice the pre-mRNA to remove introns (and some exons) - leaving only useful sections of exons

this is done by an enzyme called a spliceosome (this cuts, sticks and glues DNA)

now mature/functional mRNA can leave nucleus

Question 21

What are isoforms?

Answer 21

isoforms are different arrangements of exons- therefore one gene can produce several polypeptides because different exons can be removed during splicing.

Question 22

define epigenetics

Answer 22

the study of how your genome is expressed

Question 23

explain DNA methylation

Answer 23

happens to adenine or cytosine (cytosine in vertebrates)

done by enzyme methyltransferase

• CH3 (methyl) group added to the pyrimidine (in nitrogenous base)
• this can only happen at a CpG region (when a C and G are next to each other)

-occurs at promotor regions and prevents activation of RNA polymerase , so the gene is not transcribed

Question 24

how can methylation change your gene over time?

Answer 24

although your genome (DNA) does not change , your epigenome does vary.

more or less (demethylation) methyl groups are added during different stages of life.

they are hereditary

your epigenome can also be affected by factors of the environment , such as diet and smoking

Question 25

explain histone modification

Answer 25

histone and DNA is chromatin. the histone can either be: acetylated: a CH3 joins to lycine or leucine acetyl group transferred to molecules (donated by acetyl coenzyme A) activates chromatin, allowing genes to be transcribed methylated: a CH3 joins to lycine or leucine this tightens DNA, having an inhibitory effect

Question 26

stem cells

Answer 26

are capable of self renewal and of producing new cells that differentiate into other specialised types of cell

Question 27

define totipotent , pluripotent and multipotent cells

Answer 27

all are unspecialised and can keep dividing. toti - can differentiate into any cell type, including placenta pluri- can differentiate into any cell , apart from placenta multi- can only differentiate into cells of a certain tissue

Question 28

pluripotent cells are affected by _________________ within blastocyst ?

Answer 28

location

Question 29

what are induced pluripotent stem cells? (iPS cells)

Answer 29

adult stem cells taken from the body that have been reprogrammed to become pluripotent again. 1) fibroblasts (connective tissue) taken from skin samples. 2) transcription factors added to activate specific genes. 3) iPS behave similarly to embryonic stem cells.

Question 30

how is therapeutic cloning carried out?

Answer 30

remove nucleus from both body cell and ovum combine body cell nucleus into ovum multiply to make more pluripotent cells separate and allow them to differentiate transport into patient with no rejection issues.

Question 31

How is recombinant DNA made? (DNA from more than one organism)

Answer 31

1) isolate the gene method 1: use restriction endonucleases which cuts a specific base sequence of DNA. Leaves 'sticky ends', exposed complementary bases that can clip back together method 2: use reverse transcriptase to turn mRNA into cDNA (complimentary DNA) DNA polymerase used to make the other strand of DNA 2) cut plasmid with same restriction endonuclease to leave complementary sticky ends. 3) join plasmid and gene with DNA ligase 4)reincorporate plasmid into host nucleus NEXT (after either method) plasmids are vector for gene to be inserted into another organism plasmid's sticky ends are complimentary to sticky ends of DNA plasmid is cut with restriction endonuclease enzyme and DNA ligase sticks DNA into plasmid by joining up sugar phosphate backbone. Reincorporate plasmid into host cell nucleus.

Question 32

What is the method of identifying recombiant DNA?

Answer 32

REPLICA PLATING plasmid contains marker gene (these could be fluorescent, antibiotic resistant or have specific requirement for nutrient) grow bacteria on plate that contains everything (master plate) sample bacteria using velvet grow again without the factor the plasmid should contain compare to the original plate to compare which colonies are modified

Question 33

What are the uses of recombiant DNA?

Answer 33

identify what a particular gene does create many copies of a particular protein insert useful genes into an organism so it expresses a particular protein.

Question 34

What are the four methods of inserting DNA into cells?

Answer 34

1) gene gun coat recombinant DNA in heavy metal and fire through membrane. 2)use virus insert weakened form of virus into recombinant DNA cells (endocytosis) 3)liposome wrapping lipid droplets fuse with cell surface membrane and can therefore enter cell 4)microinjection insert DNA directly into nucleus

Question 35

What are knockout mice?

Answer 35

knockout mice are mice with one or more genes silenced , via the insertion of a similar gene which make the original gene impossible to read. they can be used to investigate gene function or understand disease. 1) insert gene similar to one being investigated 2)this stops original gene being transcribed 3)insert into embryonic stem cell (zygote) and then into a host mother. 4)chimeric mice are born- they have both versions of gene 5) cross breeding with normal mouse leads to homozygous mice which only have the knockout gene.

Question 36

define transgenic plants

Answer 36

these are plants which contain genetic material from an unrelated organism.

Question 37

How are soya beans transgenic?

Answer 37

Linoleic acid is replaced by oleic acid which oxidises less easily and therefore doesn't go off as quickly.

Question 38

What can genetically modified plants be used for?

Answer 38

pesticide protection changing nutrient value of plants increase crop yield

Question 39

What are the objections to genetically modified crops?

Answer 39

favours richer, more developed countries unnatural

Question 40

What is DNA sequencing ?

Answer 40

DNA sequencing is used to predict amino acid sequence of proteins and determine links to genetic conditions.

Question 41

What is DNA profiling and how does it work?

Answer 41

DNA profiling is used to identify criminals and test paternity. 1) fragments are cut with restriction endonuclease enzymes either side of the satellite. 2) fragments separated and visualised using gel electrophoresis. 3) southern blotting alkaline buffer added which denatures the DNA so it becomes single stranded. Nylon filter draws the DNA fragments leaving them as 'blots' attached. 4) gene probes (short single strands of DNA that have sequences complementary to sequences being sought) have fluorescent markers and bind with complementary DNA strands in a process called hybridisation. 5) graph the data collected from the probe.

Question 42

What are the 3 ways gene expression can be changed by epigenetics?

Answer 42

DNA methylation Histone modification Non-coding RNA

Question 43

Why are epigenetic modifications important?

Answer 43

ensure cell differentiation

Question 44

evaluate induced pluripotent stem cells

Answer 44

+ overcomes ethical issues as a zygote is not made - have shown tendency to be cancerous

Question 45

What are gene markers?

Answer 45

gene markers are used to show where a foreign gene has been inserted. Fluorescence, antibiotic resistance combined with replica plating can be used as gene markers.

Question 46

What is replica plating?

Answer 46

used to access if gene was inserted correctly when creating recombinant DNA. plasmid contains marker gene. bacteria grown on master plate. sample bacteria using velvet. grow again without the factor the plasmid should contain, compare to the original plate to identify which colonies are modified.